Multiple drug resistance gene atrD of aspergillus nidulans

ABSTRACT

The invention provides isolated nucleic acid compounds encoding a multiple drug resistance protein of  Aspergillus nidulans.  Vectors and transformed host cells comprising the multiple drug resistance-encoding DNA of  Aspergillus nidulans  atrD are also provided. The invention further provides assays which utilize these transformed host cells.

TECHNICAL FIELD OF THE INVENTION

[0001] This invention relates to recombinant DNA technology. In particular, the invention concerns the cloning of nucleic acid encoding a multiple drug resistance protein of Aspergillus nidulans.

BACKGROUND OF THE INVENTION

[0002] Multiple drug resistance (MDR) mediated by the human mdr-1 gene product was initially recognized during the course of developing regimens for cancer chemotherapy (Fojo et al., 1987, Journal of Clinical Oncology 5:1922-1927). A multiple drug resistant cancer cell line exhibits resistance to high levels of a large variety of cytotoxic compounds. Frequently these cytotoxic compounds will have no common structural features nor will they interact with a common target within the cell. Resistance to these cytotoxic agents is mediated by an outward directed, ATP-dependent pump encoded by the mdr-1 gene. By this mechanism, toxic levels of a particular cytotoxic compound are not allowed to accumulate within the cell.

[0003] MDR-like genes have been identified in a number of divergent organisms including numerous bacterial species, the fruit fly Drosophila melanogaster, Plasmodium falciparum, the yeast Saccharomyces cerevisiae, Caenorhabditis elegans, Leishmania donovanii, marine sponges, the plant Arabidopsis thaliana, as well as Homo sapiens. Extensive searches have revealed several classes of compounds that are able to reverse the MDR phenotype of multiple drug resistant human cancer cell lines rendering them susceptible to the effects of cytotoxic compounds. These compounds, referred to herein as “MDR inhibitors”, include for example, calcium channel blockers, antiarrhythmics, antihypertensives, antibiotics, antihistamines, immuno-suppressants, steroid hormones, modified steroids, lipophilic cations, diterpenes, detergents, antidepressants, and antipsychotics (Gottesman and Pastan, 1993, Annual Review of Biochemistry 62:385-427). Clinical application of human MDR inhibitors to cancer chemotherapy has become an area of intensive focus for research.

[0004] On another front, the discovery and development of antifungal compounds for specific fungal species has also met with some degree of success. Candida species represent the majority of fungal infections, and screens for new antifungal compounds have been designed to discover anti-Candida compounds. During development of antifungal agents, activity has generally been optimized based on activity against Candida albicans. As a consequence, these anti-Candida compounds frequently do not possess clinically significant activity against other fungal species such as Aspergillus nidulans. However, it is interesting to note that at higher concentrations some anti-Candida compounds are able to kill other fungal species such as A. nidulans and A. fumigatus. This type of observation suggests that the antifungal target(s) of these anti-Candida compounds is present in A. nidulans as well. Such results indicate that A. nidulans may possess a natural mechanism of resistance that permits them to survive in clinically relevant concentrations of antifungal compounds. Until the present invention, such a general mechanism of resistance to antifungal compounds in A. nidulans has remained undescribed.

SUMMARY OF THE INVENTION

[0005] The invention provides, inter alia, isolated nucleic acid molecules that comprise nucleic acid encoding a multiple drug resistance protein from Aspergillus nidulans, herein referred to as atrD, vectors encoding atrD, and host cells transformed with these vectors.

[0006] In another embodiment, the invention provides a method for determining the fungal MDR inhibition activity of a compound which comprises:

[0007] a) placing a culture of fungal cells, transformed with a vector capable of expressing atrD, in the presence of:

[0008] (i) an antifungal agent to which said fungal cell is resistant, but to which said fungal cell is sensitive in its untransformed state;

[0009] (ii) a compound suspected of possessing fungal MDR inhibition activity; and

[0010] b) determining the fungal MDR inhibition activity of said compound by measuring the ability of the antifungal agent to inhibit the growth of said fungal cell.

[0011] In still another embodiment the present invention relates to strains of A. nidulans in which the atrD gene is disrupted or otherwise mutated such that the atrD protein is not produced in said strains.

[0012] In yet another embodiment, the present invention relates to a method for identifiying new antifungal compounds comprising the use of atrD gene disruption or gene replacement strains of A. nidulans.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention provides isolated nucleic acid molecules that comprise a nucleic acid sequence encoding atrD. The cDNA (complementary deoxyribonucleic acid) sequence encoding atrD is provided in the Sequence Listing as SEQ ID NO: 1. The amino acid sequence of the protein encoded by atrD is provided in the Sequence Listing as SEQ ID NO: 2.

[0014] Those skilled in the art will recognize that the degenerate nature of the genetic code enables one to construct many different nucleic acid sequences that encode the amino acid sequence of SEQ ID NO: 2. The cDNA sequence depicted by SEQ ID NO: 1 is only one of many possible atrD-encoding sequences. Consequently, the constructions described below and in the accompanying examples for the preferred nucleic acid molecules, vectors, and transformants of the invention are illustrative and are not intended to limit the scope of the invention.

[0015] All nucleotide and amino acid abbreviations used in this disclosure are those accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. §1.822(b)(1994).

[0016] The term “vector” refers to any autonomously replicating or integrating agent, including but not limited to plasmids, cosmids, and viruses (including phage), comprising a nucleic acid molecule to which one or more additional nucleic acid molecules can be added. Included in the definition of “vector” is the term “expression vector”. Vectors are used either to amplify and/or to express deoxyribonucleic acid (DNA), either genomic or cDNA, or RNA (ribonucleic acid) which encodes atrD, or to amplify DNA or RNA that hybridizes with DNA or RNA encoding atrD.

[0017] The term “expression vector” refers to vectors which comprise a transcriptional promoter (hereinafter “promoter”) and other regulatory sequences positioned to drive expression of a DNA segment that encodes atrD. Expression vectors of the present invention are replicable DNA constructs in which a DNA sequence encoding atrD is operably linked to suitable control sequences capable of effecting the expression of atrD in a suitable host. Such control sequences include a promoter, an optional operator sequence to control transcription, a sequence encoding suitable MRNA ribosomal binding sites, and sequences which control termination of transcription and translation. DNA regions are operably linked when they are functionally related to each other. For example, a promoter is operably linked to a DNA coding sequence if it controls the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

[0018] The term “MDR inhibition activity” refers to the ability of a compound to inhibit the MDR activity of a host cell, thereby increasing the antifungal activity of an antifungal compound against said host cell.

[0019] In the present invention, atrD may be synthesized by host cells transformed with vectors that provide for the expression of DNA encoding atrD. The DNA encoding atrD may be the natural sequence or a synthetic sequence or a combination of both (“semi-synthetic sequence”). The in vitro or in vivo transcription and translation of these sequences results in the production of atrD. Synthetic and semi-synthetic sequences encoding atrD may be constructed by techniques well known in the art. See Brown et al. (1979) Methods in Enzymology, Academic Press, N.Y., 68:109-151. atrD-encoding DNA, or portions thereof, may be generated using a conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A,380B, 394 or 3948 DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404).

[0020] Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of nucleic acid sequences may be constructed which encode atrD. All such nucleic acid sequences are provided by the present invention. These sequences can be prepared by a variety of methods and, therefore, the invention is not limited to any particular preparation means. The nucleic acid sequences of the invention can be produced by a number of procedures, including DNA synthesis, cDNA cloning, genomic cloning, polymerase chain reaction (PCR) technology, or a combination of these approaches. These and other techniques are described by Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), or Current Protocols in Molecular Biology (F. M. Ausubel et al., 1989 and supplements). The contents of both of these references are incorporated herein by reference.

[0021] In another aspect, this invention provides the cDNA encoding atrD, which may be obtained by synthesizing the desired portion of SEQ ID NO:1 or by following the procedure carried out by Applicants. This procedure involved construction of a cosmid genomic DNA library from Aspergillus nidulans strain OC-1, a mutant derived from A42355. This library was screened for genes related to MDRs using a homologous probe generated by PCR. Degenerate PCR primers directed towards amplification of DNA sequences encoding highly conserved regions found in the ATP-binding domain of several MDR genes were synthesized. PCR using these primers and Aspergillus nidulans genomic DNA as template produced an approximately 400 base pair DNA fragment. The DNA sequence of this fragment was highly homologous to the ATP-binding region of several MDRs as predicted. This fragment was used as a hybridization probe to identify cosmid clones containing the entire atrD gene. A subclone from one such cosmid containing the entire atrD gene was sequenced to ascertain the entire sequence of atrD.

[0022] To effect the translation of atrD-encoding mRNA, one inserts the natural, synthetic, or semi-synthetic atrD-encoding DNA sequence into any of a large number of appropriate expression vectors through the use of appropriate restriction endonucleases and DNA ligases. Synthetic and semi-synthetic atrD-encoding DNA sequences can be designed, and natural atrD-encoding nucleic acid can be modified, to possess restriction endonuclease cleavage sites to facilitate isolation from and integration into these vectors. Particular restriction endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the expression vector utilized. Restriction enzyme sites are chosen so as to properly orient the atrD-encoding DNA with the control sequences to achieve proper in-frame transcription and translation of the atrD molecule. The atrD-encoding DNA must be positioned so as to be in proper reading frame with the promoter and ribosome binding site of the expression vector, both of which are functional in the host cell in which atrD is to be expressed.

[0023] Expression of atrD in fungal cells, such as Saccharomyces cerevisiae is preferred. Suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (found on plasmid pAP12BD (ATCC 53231) and described in U.S. Pat. No. 4,935,350, Jun. 19, 1990) or other glycolytic enzymes such as enolase (found on plasmid pAC1 (ATCC 39532)), glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPC1 (ATCC 57090, 57091)), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Inducible yeast promoters have the additional advantage of transcription controlled by growth conditions. Such promoters include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphotase, degradative enzymes associated with nitrogen metabolism, metallothionein (contained on plasmid vector pCL28XhoLHBPV (ATCC 39475), U.S. Pat. No. 4,840,896), glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization (GAL1 found on plasmid pRY121 (ATCC 37658) and on plasmid pPST5, described below). Suitable vectors and promoters for use in yeast expression are further described by R. Hitzeman et al., in European Patent Publication No. 73,657A. Yeast enhancers such as the UAS Gal enhancer from Saccharomyces cerevisiae (found in conjunction with the CYCI promoter on plasmid YEpsec—hI1beta, ATCC 67024), also are advantageously used with yeast promoters.

[0024] A variety of expression vectors useful in the present invention are well known in the art. For expression in Saccharomyces, the plasmid YRp7, for example, (ATCC-40053, Stinchcomb et al., 1979, Nature 282:39; Kingsman et al., 1979, Gene 7:141 ; Tschemper et al., 1980, Gene 10:157) is commonly used. This plasmid contains the trp gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC 44076 or PEP4-1 (Jones, 1977, Genetics 85:12).

[0025] Expression vectors useful in the expression of atrD can be constructed by a number of methods. For example, the cDNA sequence encoding atrD can be synthesized using DNA synthesis techniques such as those described above. Such synthetic DNA can be synthesized to contain cohesive ends that allow facile cloning into an appropriately digested expression vector. For example, the cDNA encoding atrD can be synthesized to contain NotI cohesive ends. Such a synthetic DNA fragment can be ligated into a NotI-digested expression vector such as pYES-2 (Invitrogen Corp., San Diego Calif. 92121).

[0026] An expression vector can also be constructed in the following manner. Logarithmic phase Aspergillus nidulans cells are disrupted by grinding under liquid nitrogen according to the procedure of Minuth et al., 1982 (Current Genetics 5:227-231). Aspergillus nidulans mRNA is preferably isolated from the disrupted cells using the QuickPrep® mRNA Purification Kit (Pharmacia Biotech) according to the instructions of the manufacturer. cDNA is produced from the isolated mRNA using the TimeSaver® cDNA Synthesis Kit (Pharmacia Biotech) using oligo (dT) according to the procedure described by the manufacturer. In this process an EcoRI/NotI adapter (Stratagene, Inc.) is ligated to each end of the double stranded cDNA. The adapter modified cDNA is ligated into the vector Lambda Zap^(R)II® using the Predigested Lambda Zap^(R)II®/EcoRI/CIAP Cloning Kit (Stratagene, Inc.) according to the instructions of the manufacturer to create a cDNA library.

[0027] The library is screened for full-length cDNA encoding atrD using a ³²P-radiolabeled fragment of the atrD gene. In this manner, a full-length cDNA clone is recovered from the Aspergillus nidulans cDNA library. A full-length cDNA clone recovered from the library is removed from the Lambda Zap^(R)II® vector by digestion with the restriction endonuclease NotI which produces a DNA fragment encoding atrD. The atrD encoding fragment is subcloned into plasmid pYES2 for expression studies. In this plasmid the atrD gene is operably linked to the Saccharomyces cerevisiae GAL1 promoter at the 5′ end, and the yeast cycl transcription terminator at the 3′ end. This plasmid further comprises the ColE1 origin of replication (ColE1) which allows replication in Escherichia coli host cells, and the ampicillin resistance gene (Amp) for selection of E. coli cells transformed with the plasmid grown in the presence of ampicillin. The expression plasmid further comprises the yeast 2 μ origin of replication (2 μ ori) allowing replication in yeast host cells, the yeast URA3 gene for selection of S. cerevisiae cells transformed with the plasmid grown in a medium lacking uracil, and the origin of replication from the f1 filamentous phage.

[0028] In a preferred embodiment of the invention Saccharomyces cerevisiae INVSc1 or INVSc2 cells (Invitrogen Corp., Sorrento Valley Blvd., San Diego Calif. 92121) are employed as host cells, but numerous other cell lines are available for this use. The transformed host cells are plated on an appropriate medium under selective pressure (minimal medium lacking uracil). The cultures are then incubated for a time and temperature appropriate to the host cell line employed.

[0029] The techniques involved in the transformation of yeast cells such as Saccharomyces cerevisiae cells are well known in the art and may be found in such general references as Ausubel et al., Current Protocols in Molecular Biology (1989), John Wiley & Sons, New York, N.Y. and supplements. The precise conditions under which the transformed yeast cells are cultured is dependent upon the nature of the yeast host cell line and the vectors employed.

[0030] Nucleic acid, either RNA or DNA, which encodes atrD, or a portion thereof, is also useful in producing nucleic acid molecules useful in diagnostic assays for the detection of atrD MRNA, atrD cDNA, or atrD genomic DNA. Further, nucleic acid, either RNA or DNA, which does not encode atrD, but which nonetheless is capable of hybridizing with atrD-encoding DNA or RNA is also useful in such diagnostic assays. These nucleic acid molecules may be covalently labeled by known methods with a detectable moiety such as a fluorescent group, a radioactive atom or a chemiluminescent group. The labeled nucleic acid is then used in conventional hybridization assays, such as Southern or Northern hybridization assays, or polymerase chain reaction assays (PCR), to identify hybridizing DNA, cDNA, or RNA molecules. PCR assays may also be performed using unlabeled nucleic acid molecules. Such assays may be employed to identify atrD vectors and transformants and in in vitro diagnosis to detect atrD-like mRNA, CDNA, or genomic DNA from other organisms.

[0031] U.S. Pat. application Ser. No. 08/111680, the entire contents of which are hereby incorporated herein by reference, describes the use of combination therapy involving an antifungal agent possessing a proven spectrum of activity, with a fungal MDR inhibitor to treat fungal infections. This combination therapy approach enables an extension of the spectrum of antifungal activity for a given antifungal compound which previously had only demonstrated limited clinically relevant antifungal activity. Similarly, compounds with demonstrated antifungal activity can also be potentiated by a fungal MDR inhibitor such that the antifungal activity of these compounds is extended to previously resistant species. To identify compounds useful in such combination therapy the present invention provides an assay method for identifying compounds with Aspergillus nidulans MDR inhibition activity. Host cells that express atrD provide an excellent means for the identification of compounds useful as inhibitors of Aspergillus nidulans MDR activity. Generally, the assay utilizes a culture of a yeast cell transformed with a vector which provides expression of atrD. The expression of atrD by the host cell enables the host cell to grow in the presence of an antifungal compound to which the yeast cell is sensitive to in the untransformed state. Thus, the transformed yeast cell culture is grown in the presence of i) an antifungal agent to which the untransformed yeast cell is sensitive, but to which the transformed host cell is resistant, and ii) a compound that is suspected of being an MDR inhibitor. The effect of the suspected MDR inhibitor is measured by testing for the ability of the antifungal compound to inhibit the growth of the transformed yeast cell. Such inhibition will occur if the suspected Aspergillus nidulans MDR inhibitor blocks the ability of atrD to prevent the antifungal compound from acting on the yeast cell. An illustrative example of such an assay is provided in Example 3.

[0032] In order to illustrate more fully the operation of this invention, the following examples are provided, but are not to be construed as a limitation on the scope of the invention.

EXAMPLE 1 Source of the atrD-Encoding Genomic DNA and cDNA of Aspergillus nidulans

[0033] Genomic DNA encoding atrD, or the corresponding cDNA sequence (presented in SEQ ID NO:1), may be from a natural sequence, a synthetic source or a combination of both (“semi-synthetic sequence”). The in vitro or in vivo transcription and translation of these sequences results in the production of atrD. Synthetic and semi-synthetic sequences encoding atrD may be constructed by techniques well known in the art. See Brown et al. (1979) Methods in Enzymology, Academic Press, N.Y., 68:109-151. atrD-encoding DNA, or portions thereof, may be generated using a conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A, 380B, 384 or 3848 DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404). The polymerase chain reaction is especially useful in generating these DNA sequences. PCR primers are constructed which include the translational start (ATG) and translational stop codon (TAG) of atrD. Restriction enzyme sites may be included on these PCR primers outside of the atrD coding region to facilitate rapid cloning into expression vectors. Aspergillus nidulans genomic DNA is used as the PCR template for synthesis of atrD including introns which is useful for expression studies in closely related fungi. In contrast, cDNA is used as the PCR template for synthesis of atrD devoid of introns which is useful for expression in foreign hosts such as lSaccharomyces cerevisiae or bacterial hosts such as Escherichia coli.

[0034] EXAMPLE 2

Expression of the atrD Protein

[0035]Saccharomyces cerevisiae INVSc1 cells (Invitrogen Corp., San Diego Calif. 92191) are transformed with the plasmid containing atrD by the technique described by J. D. Beggs, 1988, Nature 275:104-109). The transformed yeast cells are grown in a broth medium containing YNB/CSM-Ura/raf (YNB/CSM-Ura [Yeast Nitrogen Base (Difco Laboratories, Detroit, Mich.) supplemented with CSM-URA (Bio 101, Inc.)] supplemented with 4% raffinose) at 28° C. in a shaker incubator until the culture is saturated. To induce expression of atrD, a portion of the culture is used to inoculate a flask containing YNB/CSM-Ura medium supplemented with 2% galactose (YNB/CSM-Ura/gal) rather than raffinose as the sole carbon source. The inoculated flask is incubated at 28° C. for about 16 hours.

EXAMPLE 3 Antifungal Potentiator Assay

[0036] Approximately 1×10⁶ cells of a Saccharomyces cerevisiae INVSc1 culture expressing atrD are delivered to each of several agar plates containing YNB/CSM-Ura/gal. The agar surface is allowed to dry in a biohazard hood.

[0037] An antifungal compound that the untransformed yeast cell is typically sensitive to is dissolved in an appropriate solvent at a concentration that is biologically effective. Twenty μl of the solution is delivered to an antibiotic susceptibility test disc (Difco Laboratories, Detroit, Mich.). After addition of the antifungal solution the disc is allowed to air dry in a biohazard hood. When dry, the disc is placed on the surface of the petri plates containing the transformed Saccharomyces cerevisiae INVSc1 cells.

[0038] Compounds to be tested for the ability to inhibit atrD are dissolved in dimethylsulfoxide (DMSO). The amount of compound added to the DMSO depends on the solubility of the individual compound to be tested. Twenty μl of the suspensions containing a compound to be tested are delivered to an antibiotic susceptibility test disc (Difco Laboratories, Detroit, Mich.). The disc is then placed on the surface of the dried petri plates containing the transformed Saccharomyces cerevisiae INVSc1 cells approximately 2 cm from the antifungal-containing disc. Petri plates containing the two discs are incubated at 28° C. for about 16-48 hours.

[0039] Following this incubation period, the petri plates are examined for zones of growth inhibition around the discs. A zone of growth inhibition near the antifungal disc on the test plate indicates that the compound being tested for MDR inhibition activity blocks the activity of atrD and allows the antifungal compound to inhibit the growth of the yeast host cell. Such compounds are said to possess MDR inhibition activity. Little or no zone of growth inhibition indicates that the test compound does not block MDR activity and, thus, atrD is allowed to act upon the antifungal compound to prevent its activity upon the host cell.

EXAMPLE 4 Screen For Novel Antifungal Compounds

[0040] A plasmid molecule is constructed which contains DNA sequence information required for replication and genetic transformation in E. coli (e.g. ampicillin resistance). The plasmid also comprises DNA sequences encoding a marker for selection in fungal cells (e.g. hygromycin B phosphotransferase, phleomycin resistance, G418 resistance) under the control of an A. nidulans promoter. Additionally, the plasmid contains an internal portion of the atrD gene (e.g. about 3000 base pairs which lack 500 base pairs at the N-terminal end, and about 500 base pairs at the C-terminal end of the coding region specified by SEQ ID NO:1). The atrD gene fragment enables a single crossover gene disruption when transformed or otherwise introduced into A. nidulans.

[0041] Alternatively, a 5 kilobase pair to 6 kilobase pair region of A. nidulans genomic DNA containing the atrD gene is subcloned into the aforementioned plasmid. Then, a central portion of the atrD gene is removed and replaced with a selectable marker, such as hyromycin B phosphotransferase, for a double crossover gene replacement.

[0042] Gene disruption and gene replacement procedures for A. nidulans are well known in the art (See e.g. May et al, J. Cell Biol. 101, 712, 1985; Jones and Sealy-Lewis, Curr. Genet. 17, 81, 1990). Transformants are recovered on an appropriate selection medium, for example, hygromycin (if hygromycin B gene is used in the construction of disruption cassette). Gene replacement, or gene disruption, is verified by any suitable method, for example, by Southern blot hybridization.

[0043] Gene disruption or gene replacement strains are rendered hypersensitive to antifungal compounds, and are useful in screens for new antifungal compounds in whole cell growth inhibition studies.

1 3 4002 base pairs nucleic acid single linear cDNA NO NO CDS 1..4002 1 ATG TCC CCG CTA GAG ACA AAT CCC CTT TCG CCA GAG ACT GCT ATG CGC 48 Met Ser Pro Leu Glu Thr Asn Pro Leu Ser Pro Glu Thr Ala Met Arg 1 5 10 15 GAA CCT GCT GAG ACT TCA ACG ACG GAG GAG CAA GCT TCT ACA CCA CAC 96 Glu Pro Ala Glu Thr Ser Thr Thr Glu Glu Gln Ala Ser Thr Pro His 20 25 30 GCT GCG GAC GAG AAG AAA ATC CTC AGC GAC CTC TCG GCT CCA TCT AGT 144 Ala Ala Asp Glu Lys Lys Ile Leu Ser Asp Leu Ser Ala Pro Ser Ser 35 40 45 ACT ACA GCA ACC CCC GCA GAC AAG GAG CAC CGT CCT AAA TCG TCG TCC 192 Thr Thr Ala Thr Pro Ala Asp Lys Glu His Arg Pro Lys Ser Ser Ser 50 55 60 AGC AAT AAT GCG GTC TCG GTC AAC GAA GTC GAT GCG CTT ATT GCG CAC 240 Ser Asn Asn Ala Val Ser Val Asn Glu Val Asp Ala Leu Ile Ala His 65 70 75 80 CTG CCA GAA GAC GAG AGG CAG GTC TTG AAG ACG CAG CTG GAG GAG ATC 288 Leu Pro Glu Asp Glu Arg Gln Val Leu Lys Thr Gln Leu Glu Glu Ile 85 90 95 AAA GTA AAC ATC TCC TTC TTC GGT CTC TGG CGG TAT GCA ACA AAG ATG 336 Lys Val Asn Ile Ser Phe Phe Gly Leu Trp Arg Tyr Ala Thr Lys Met 100 105 110 GAT ATA CTT ATC ATG GTA ATC AGT ACA ATC TGT GCC ATT GCT GCC GCG 384 Asp Ile Leu Ile Met Val Ile Ser Thr Ile Cys Ala Ile Ala Ala Ala 115 120 125 TCG ACT TTC CAG AGG ATA ATG TTA TAT CAA ATC TCG TAC GAC GAG TTC 432 Ser Thr Phe Gln Arg Ile Met Leu Tyr Gln Ile Ser Tyr Asp Glu Phe 130 135 140 TAT GAT GAA TTG ACC AAG AAC GTA CTG TAC TTC GTA TAC CTC GGT ATC 480 Tyr Asp Glu Leu Thr Lys Asn Val Leu Tyr Phe Val Tyr Leu Gly Ile 145 150 155 160 GGC GAG TTT GTC ACT GTC TAT GTT AGT ACT GTT GGC TTC ATC TAT ACC 528 Gly Glu Phe Val Thr Val Tyr Val Ser Thr Val Gly Phe Ile Tyr Thr 165 170 175 GGA GAA CAC GCC ACG CAG AAG ATC CGC GAG TAT TAC CTT GAG TCT ATC 576 Gly Glu His Ala Thr Gln Lys Ile Arg Glu Tyr Tyr Leu Glu Ser Ile 180 185 190 CTG CGC CAG AAC ATT GGC TAT TTT GAT AAA CTC GGT GCC GGG GAA GTG 624 Leu Arg Gln Asn Ile Gly Tyr Phe Asp Lys Leu Gly Ala Gly Glu Val 195 200 205 ACC ACC CGT ATA ACA GCC GAT ACA AAC CTT ATC CAG GAT GGC ATT TCG 672 Thr Thr Arg Ile Thr Ala Asp Thr Asn Leu Ile Gln Asp Gly Ile Ser 210 215 220 GAG AAG GTC GGT CTC ACT TTG ACT GCC CTG GCG ACA TTC GTG ACA GCA 720 Glu Lys Val Gly Leu Thr Leu Thr Ala Leu Ala Thr Phe Val Thr Ala 225 230 235 240 TTC ATT ATC GCC TAC GTC AAA TAC TGG AAG TTG GCT CTA ATT TGC AGC 768 Phe Ile Ile Ala Tyr Val Lys Tyr Trp Lys Leu Ala Leu Ile Cys Ser 245 250 255 TCA ACA ATT GTG GCC CTC GTT CTC ACC ATG GGC GGT GGT TCT CAG TTT 816 Ser Thr Ile Val Ala Leu Val Leu Thr Met Gly Gly Gly Ser Gln Phe 260 265 270 ATC ATC AAG TAC AGC AAA AAG TCG CTT GAC AGC TAC GGT GCA GGC GGC 864 Ile Ile Lys Tyr Ser Lys Lys Ser Leu Asp Ser Tyr Gly Ala Gly Gly 275 280 285 ACT GTT GCG GAA GAG GTC ATC AGC TCC ATC AGA AAT GCC ACA GCG TTT 912 Thr Val Ala Glu Glu Val Ile Ser Ser Ile Arg Asn Ala Thr Ala Phe 290 295 300 GGC ACC CAA GAC AAG CTT GCG AAG CAG TAT GAG GTC CAC TTA GAC GAA 960 Gly Thr Gln Asp Lys Leu Ala Lys Gln Tyr Glu Val His Leu Asp Glu 305 310 315 320 GCT GAG AAA TGG GGA ACA AAG AAC CAG ATT GTC ATG GGT TTC ATG ATT 1008 Ala Glu Lys Trp Gly Thr Lys Asn Gln Ile Val Met Gly Phe Met Ile 325 330 335 GGC GCC ATG TTT GGC CTT ATG TAC TCG AAC TAC GGT CTT GGC TTC TGG 1056 Gly Ala Met Phe Gly Leu Met Tyr Ser Asn Tyr Gly Leu Gly Phe Trp 340 345 350 ATG GGT TCT CGT TTC CTG GTA GAT GGT GCA GTC GAT GTG GGT GAT ATT 1104 Met Gly Ser Arg Phe Leu Val Asp Gly Ala Val Asp Val Gly Asp Ile 355 360 365 CTC ACA GTT CTC ATG GCC ATC TTG ATC GGA TCG TTC TCC TTG GGG AAC 1152 Leu Thr Val Leu Met Ala Ile Leu Ile Gly Ser Phe Ser Leu Gly Asn 370 375 380 GTT AGT CCA AAT GCT CAA GCA TTT ACA AAC GCT GTG GCC GCG GCC GCA 1200 Val Ser Pro Asn Ala Gln Ala Phe Thr Asn Ala Val Ala Ala Ala Ala 385 390 395 400 AAG ATA TTT GGA ACG ATC GAT CGC CAG TCC CCA TTA GAT CCA TAT TCG 1248 Lys Ile Phe Gly Thr Ile Asp Arg Gln Ser Pro Leu Asp Pro Tyr Ser 405 410 415 AAC GAA GGG AAG ACG CTC GAC CAT TTT GAG GGC CAC ATT GAG TTA CGC 1296 Asn Glu Gly Lys Thr Leu Asp His Phe Glu Gly His Ile Glu Leu Arg 420 425 430 AAT GTC AAG CAT ATT TAC CCA TCT AGA CCC GAG GTC ACC GTC ATG GAG 1344 Asn Val Lys His Ile Tyr Pro Ser Arg Pro Glu Val Thr Val Met Glu 435 440 445 GAT GTT TCT CTG TCA ATG CCC GCT GGA AAA ACA ACC GCT TTA GTC GGC 1392 Asp Val Ser Leu Ser Met Pro Ala Gly Lys Thr Thr Ala Leu Val Gly 450 455 460 CCC TCT GGC TCT GGA AAA AGT ACG GTG GTC GGC TTG GTT GAG CGA TTC 1440 Pro Ser Gly Ser Gly Lys Ser Thr Val Val Gly Leu Val Glu Arg Phe 465 470 475 480 TAC ATG CCT GTT CGC GGT ACG GTT TTG CTG GAT GGC CAT GAC ATC AAG 1488 Tyr Met Pro Val Arg Gly Thr Val Leu Leu Asp Gly His Asp Ile Lys 485 490 495 GAC CTC AAT CTC CGC TGG CTT CGC CAA CAG ATC TCT TTG GTT AGC CAG 1536 Asp Leu Asn Leu Arg Trp Leu Arg Gln Gln Ile Ser Leu Val Ser Gln 500 505 510 GAG CCT GTT CTT TTT GGC ACG ACG ATT TAT AAG AAT ATT AGG CAC GGT 1584 Glu Pro Val Leu Phe Gly Thr Thr Ile Tyr Lys Asn Ile Arg His Gly 515 520 525 CTC ATC GGC ACA AAG TAC GAG AAT GAA TCC GAG GAT AAG GTC CGG GAA 1632 Leu Ile Gly Thr Lys Tyr Glu Asn Glu Ser Glu Asp Lys Val Arg Glu 530 535 540 CTC ATC GAG AAC GCG GCA AAA ATG GCG AAT GCT CAT GAC TTT ATT ACT 1680 Leu Ile Glu Asn Ala Ala Lys Met Ala Asn Ala His Asp Phe Ile Thr 545 550 555 560 GCC TTG CCT GAA GGT TAT GAG ACC AAT GTT GGG CAG CGT GGC TTT CTC 1728 Ala Leu Pro Glu Gly Tyr Glu Thr Asn Val Gly Gln Arg Gly Phe Leu 565 570 575 CTT TCA GGT GGC CAG AAA CAG CGC ATT GCA ATC GCC CGT GCC GTT GTT 1776 Leu Ser Gly Gly Gln Lys Gln Arg Ile Ala Ile Ala Arg Ala Val Val 580 585 590 AGT GAC CCA AAA ATC CTG CTC CTG GAT GAA GCT ACT TCG GCC TTG GAC 1824 Ser Asp Pro Lys Ile Leu Leu Leu Asp Glu Ala Thr Ser Ala Leu Asp 595 600 605 ACA AAA TCC GAA GGC GTG GTT CAA GCA GCT TTG GAG AGG GCA GCT GAA 1872 Thr Lys Ser Glu Gly Val Val Gln Ala Ala Leu Glu Arg Ala Ala Glu 610 615 620 GGC CGA ACT ACT ATT GTG ATC GCT CAT CGC CTT TCC ACG ATC AAA ACG 1920 Gly Arg Thr Thr Ile Val Ile Ala His Arg Leu Ser Thr Ile Lys Thr 625 630 635 640 GCG CAC AAC ATT GTG GTT CTG GTC AAT GGC AAA ATT GCT GAA CAA GGA 1968 Ala His Asn Ile Val Val Leu Val Asn Gly Lys Ile Ala Glu Gln Gly 645 650 655 ACT CAC GAT GAA TTG GTT GAC CGC GGA GGC GCT TAT CGC AAA CTT GTG 2016 Thr His Asp Glu Leu Val Asp Arg Gly Gly Ala Tyr Arg Lys Leu Val 660 665 670 GAG GCT CAA CGT ATC AAT GAA CAG AAG GAA GCT GAC GCC TTG GAG GAC 2064 Glu Ala Gln Arg Ile Asn Glu Gln Lys Glu Ala Asp Ala Leu Glu Asp 675 680 685 GCC GAC GCT GAG GAT CTC ACG AAT GCA GAT ATT GCC AAA ATC AAA ACT 2112 Ala Asp Ala Glu Asp Leu Thr Asn Ala Asp Ile Ala Lys Ile Lys Thr 690 695 700 GCG TCA AGC GCA TCA TCC GAT CTC GAC GGA AAA CCC ACA ACC ATT GAC 2160 Ala Ser Ser Ala Ser Ser Asp Leu Asp Gly Lys Pro Thr Thr Ile Asp 705 710 715 720 CGC ACG GGC ACC CAC AAG TCT GTT TCC AGC GCG ATT CTT TCT AAA AGA 2208 Arg Thr Gly Thr His Lys Ser Val Ser Ser Ala Ile Leu Ser Lys Arg 725 730 735 CCC CCC GAA ACA ACT CCG AAA TAC TCA TTA TGG ACG CTG CTC AAA TTT 2256 Pro Pro Glu Thr Thr Pro Lys Tyr Ser Leu Trp Thr Leu Leu Lys Phe 740 745 750 GTT GCT TCC TTC AAC CGC CCT GAA ATC CCG TAC ATG CTC ATC GGT CTT 2304 Val Ala Ser Phe Asn Arg Pro Glu Ile Pro Tyr Met Leu Ile Gly Leu 755 760 765 GTC TTC TCA GTG TTA GCT GGT GGT GGC CAA CCC ACG CAA GCA GTG CTA 2352 Val Phe Ser Val Leu Ala Gly Gly Gly Gln Pro Thr Gln Ala Val Leu 770 775 780 TAT GCT AAA GCC ATC AGC ACA CTC TCG CTC CCA GAA TCA CAA TAT AGC 2400 Tyr Ala Lys Ala Ile Ser Thr Leu Ser Leu Pro Glu Ser Gln Tyr Ser 785 790 795 800 AAG CTT CGA CAT GAT GCG GAT TTC TGG TCA TTG ATG TTC TTC GTG GTT 2448 Lys Leu Arg His Asp Ala Asp Phe Trp Ser Leu Met Phe Phe Val Val 805 810 815 GGT ATC ATT CAG TTT ATC ACG CAG TCA ACC AAT GGT GCT GCA TTT GCC 2496 Gly Ile Ile Gln Phe Ile Thr Gln Ser Thr Asn Gly Ala Ala Phe Ala 820 825 830 GTA TGC TCC GAG AGA CTT ATT CGT CGC GCG AGA AGC ACT GCC TTT CGG 2544 Val Cys Ser Glu Arg Leu Ile Arg Arg Ala Arg Ser Thr Ala Phe Arg 835 840 845 ACG ATA CTC CGT CAA GAC ATT GCT TTC TTT GAC AAG GAA GAG AAT AGC 2592 Thr Ile Leu Arg Gln Asp Ile Ala Phe Phe Asp Lys Glu Glu Asn Ser 850 855 860 ACC GGC GCT CTG ACC TCT TTC CTG TCC ACC GAG ACG AAG CAT CTC TCC 2640 Thr Gly Ala Leu Thr Ser Phe Leu Ser Thr Glu Thr Lys His Leu Ser 865 870 875 880 GGT GTT AGC GGT GTG ACT CTA GGC ACG ATC TTG ATG ACC TCC ACG ACC 2688 Gly Val Ser Gly Val Thr Leu Gly Thr Ile Leu Met Thr Ser Thr Thr 885 890 895 CTA GGA GCG GCT ATC ATT ATT GCC CTG GCG ATT GGG TGG AAA TTG GCC 2736 Leu Gly Ala Ala Ile Ile Ile Ala Leu Ala Ile Gly Trp Lys Leu Ala 900 905 910 TTA GTT TGT ATC TCG GTT GTG CCG GTT CTC CTG GCA TGC GGT TTC TAC 2784 Leu Val Cys Ile Ser Val Val Pro Val Leu Leu Ala Cys Gly Phe Tyr 915 920 925 CGA TTC TAT ATG CTA GCC CAG TTT CAA TCA CGC TCC AAG CTT GCT TAT 2832 Arg Phe Tyr Met Leu Ala Gln Phe Gln Ser Arg Ser Lys Leu Ala Tyr 930 935 940 GAG GGA TCT GCA AAC TTT GCT TGC GAG GCT ACA TCG TCT ATC CGC ACA 2880 Glu Gly Ser Ala Asn Phe Ala Cys Glu Ala Thr Ser Ser Ile Arg Thr 945 950 955 960 GTT GCG TCA TTA ACC CGG GAA AGG GAT GTC TGG GAG ATT TAC CAT GCC 2928 Val Ala Ser Leu Thr Arg Glu Arg Asp Val Trp Glu Ile Tyr His Ala 965 970 975 CAG CTT GAC GCA CAA GGC AGG ACC AGT CTA ATC TCT GTC TTG AGG TCA 2976 Gln Leu Asp Ala Gln Gly Arg Thr Ser Leu Ile Ser Val Leu Arg Ser 980 985 990 TCC CTG TTA TAT GCG TCG TCG CAG GCA CTT GTT TTC TTC TGC GTT GCG 3024 Ser Leu Leu Tyr Ala Ser Ser Gln Ala Leu Val Phe Phe Cys Val Ala 995 1000 1005 CTC GGG TTT TGG TAC GGA GGG ACA CTT CTT GGT CAC CAC GAG TAT GAC 3072 Leu Gly Phe Trp Tyr Gly Gly Thr Leu Leu Gly His His Glu Tyr Asp 1010 1015 1020 ATT TTC CGC TTC TTT GTT TGT TTC TCC GAG ATT CTC TTT GGT GCT CAA 3120 Ile Phe Arg Phe Phe Val Cys Phe Ser Glu Ile Leu Phe Gly Ala Gln 1025 1030 1035 1040 TCC GCG GGC ACC GTC TTT TCC TTT GCA CCA GAC ATG GGC AAG GCG AAG 3168 Ser Ala Gly Thr Val Phe Ser Phe Ala Pro Asp Met Gly Lys Ala Lys 1045 1050 1055 AAT GCG GCC GCC GAA TTC CGA CGA CTG TTC GAC CGA AAG CCA CAA ATT 3216 Asn Ala Ala Ala Glu Phe Arg Arg Leu Phe Asp Arg Lys Pro Gln Ile 1060 1065 1070 GAT AAC TGG TCT GAA GAG GGC GAG AAG CTC GAA ACG GTG GAA GGT GAA 3264 Asp Asn Trp Ser Glu Glu Gly Glu Lys Leu Glu Thr Val Glu Gly Glu 1075 1080 1085 ATC GAA TTT AGG AAC GTG CAC TTC AGA TAC CCG ACC CGC CCA GAA CAG 3312 Ile Glu Phe Arg Asn Val His Phe Arg Tyr Pro Thr Arg Pro Glu Gln 1090 1095 1100 CCT GTC CTG CGC GGC TTG GAC CTG ACC GTG AAG CCT GGA CAA TAT GTT 3360 Pro Val Leu Arg Gly Leu Asp Leu Thr Val Lys Pro Gly Gln Tyr Val 1105 1110 1115 1120 GCG CTT GTC GGA CCC AGC GGT TGT GGC AAG AGT ACC ACC ATT GCA TTG 3408 Ala Leu Val Gly Pro Ser Gly Cys Gly Lys Ser Thr Thr Ile Ala Leu 1125 1130 1135 CTT GAG CGC TTT TAC GAT GCG ATT GCC GGG TCC ATC CTT GTT GAT GGG 3456 Leu Glu Arg Phe Tyr Asp Ala Ile Ala Gly Ser Ile Leu Val Asp Gly 1140 1145 1150 AAG GAC ATA AGT AAA CTA AAT ATC AAC TCC TAC CGC AGC TTT CTG TCA 3504 Lys Asp Ile Ser Lys Leu Asn Ile Asn Ser Tyr Arg Ser Phe Leu Ser 1155 1160 1165 CTG GTC AGC CAG GAG CCG ACA CTG TAC CAG GGC ACC ATC AAG GAA AAC 3552 Leu Val Ser Gln Glu Pro Thr Leu Tyr Gln Gly Thr Ile Lys Glu Asn 1170 1175 1180 ATC TTA CTT GGT ATT GTC GAA GAT GAC GTA CCG GAA GAA TTC TTG ATT 3600 Ile Leu Leu Gly Ile Val Glu Asp Asp Val Pro Glu Glu Phe Leu Ile 1185 1190 1195 1200 AAG GCT TGC AAG GAC GCT AAT ATC TAC GAC TTC ATC ATG TCG CTC CCG 3648 Lys Ala Cys Lys Asp Ala Asn Ile Tyr Asp Phe Ile Met Ser Leu Pro 1205 1210 1215 GAG GGC TTT AAT ACA GTT GTT GGC AGC AAG GGA GGC ATG TTG TCT GGC 3696 Glu Gly Phe Asn Thr Val Val Gly Ser Lys Gly Gly Met Leu Ser Gly 1220 1225 1230 GGC CAA AAG CAA CGT GTG GCC ATT GCC CGA GCC CTT CTT CGG GAT CCC 3744 Gly Gln Lys Gln Arg Val Ala Ile Ala Arg Ala Leu Leu Arg Asp Pro 1235 1240 1245 AAA ATC CTT CTT CTC GAT GAA GCG ACG TCA GCC CTC GAC TCC GAG TCA 3792 Lys Ile Leu Leu Leu Asp Glu Ala Thr Ser Ala Leu Asp Ser Glu Ser 1250 1255 1260 GAA AAG GTC GTC CAG GCG GCT TTG GAT GCC GCT GCC CGA GGC CGA ACC 3840 Glu Lys Val Val Gln Ala Ala Leu Asp Ala Ala Ala Arg Gly Arg Thr 1265 1270 1275 1280 ACA ATC GCC GTT GCA CAC CGA CTC AGC ACG ATT CAA AAG GCG GAC GTT 3888 Thr Ile Ala Val Ala His Arg Leu Ser Thr Ile Gln Lys Ala Asp Val 1285 1290 1295 ATC TAT GTT TTC GAC CAA GGC AAG ATC GTC GAA AGC GGA ACG CAC AGC 3936 Ile Tyr Val Phe Asp Gln Gly Lys Ile Val Glu Ser Gly Thr His Ser 1300 1305 1310 GAA CTG GTC CAG AAA AAG GGC CGG TAC TAC GAG CTG GTC AAC TTG CAG 3984 Glu Leu Val Gln Lys Lys Gly Arg Tyr Tyr Glu Leu Val Asn Leu Gln 1315 1320 1325 AGC TTG GGC AAG GGC CAT 4002 Ser Leu Gly Lys Gly His 1330 1334 amino acids amino acid linear protein 2 Met Ser Pro Leu Glu Thr Asn Pro Leu Ser Pro Glu Thr Ala Met Arg 1 5 10 15 Glu Pro Ala Glu Thr Ser Thr Thr Glu Glu Gln Ala Ser Thr Pro His 20 25 30 Ala Ala Asp Glu Lys Lys Ile Leu Ser Asp Leu Ser Ala Pro Ser Ser 35 40 45 Thr Thr Ala Thr Pro Ala Asp Lys Glu His Arg Pro Lys Ser Ser Ser 50 55 60 Ser Asn Asn Ala Val Ser Val Asn Glu Val Asp Ala Leu Ile Ala His 65 70 75 80 Leu Pro Glu Asp Glu Arg Gln Val Leu Lys Thr Gln Leu Glu Glu Ile 85 90 95 Lys Val Asn Ile Ser Phe Phe Gly Leu Trp Arg Tyr Ala Thr Lys Met 100 105 110 Asp Ile Leu Ile Met Val Ile Ser Thr Ile Cys Ala Ile Ala Ala Ala 115 120 125 Ser Thr Phe Gln Arg Ile Met Leu Tyr Gln Ile Ser Tyr Asp Glu Phe 130 135 140 Tyr Asp Glu Leu Thr Lys Asn Val Leu Tyr Phe Val Tyr Leu Gly Ile 145 150 155 160 Gly Glu Phe Val Thr Val Tyr Val Ser Thr Val Gly Phe Ile Tyr Thr 165 170 175 Gly Glu His Ala Thr Gln Lys Ile Arg Glu Tyr Tyr Leu Glu Ser Ile 180 185 190 Leu Arg Gln Asn Ile Gly Tyr Phe Asp Lys Leu Gly Ala Gly Glu Val 195 200 205 Thr Thr Arg Ile Thr Ala Asp Thr Asn Leu Ile Gln Asp Gly Ile Ser 210 215 220 Glu Lys Val Gly Leu Thr Leu Thr Ala Leu Ala Thr Phe Val Thr Ala 225 230 235 240 Phe Ile Ile Ala Tyr Val Lys Tyr Trp Lys Leu Ala Leu Ile Cys Ser 245 250 255 Ser Thr Ile Val Ala Leu Val Leu Thr Met Gly Gly Gly Ser Gln Phe 260 265 270 Ile Ile Lys Tyr Ser Lys Lys Ser Leu Asp Ser Tyr Gly Ala Gly Gly 275 280 285 Thr Val Ala Glu Glu Val Ile Ser Ser Ile Arg Asn Ala Thr Ala Phe 290 295 300 Gly Thr Gln Asp Lys Leu Ala Lys Gln Tyr Glu Val His Leu Asp Glu 305 310 315 320 Ala Glu Lys Trp Gly Thr Lys Asn Gln Ile Val Met Gly Phe Met Ile 325 330 335 Gly Ala Met Phe Gly Leu Met Tyr Ser Asn Tyr Gly Leu Gly Phe Trp 340 345 350 Met Gly Ser Arg Phe Leu Val Asp Gly Ala Val Asp Val Gly Asp Ile 355 360 365 Leu Thr Val Leu Met Ala Ile Leu Ile Gly Ser Phe Ser Leu Gly Asn 370 375 380 Val Ser Pro Asn Ala Gln Ala Phe Thr Asn Ala Val Ala Ala Ala Ala 385 390 395 400 Lys Ile Phe Gly Thr Ile Asp Arg Gln Ser Pro Leu Asp Pro Tyr Ser 405 410 415 Asn Glu Gly Lys Thr Leu Asp His Phe Glu Gly His Ile Glu Leu Arg 420 425 430 Asn Val Lys His Ile Tyr Pro Ser Arg Pro Glu Val Thr Val Met Glu 435 440 445 Asp Val Ser Leu Ser Met Pro Ala Gly Lys Thr Thr Ala Leu Val Gly 450 455 460 Pro Ser Gly Ser Gly Lys Ser Thr Val Val Gly Leu Val Glu Arg Phe 465 470 475 480 Tyr Met Pro Val Arg Gly Thr Val Leu Leu Asp Gly His Asp Ile Lys 485 490 495 Asp Leu Asn Leu Arg Trp Leu Arg Gln Gln Ile Ser Leu Val Ser Gln 500 505 510 Glu Pro Val Leu Phe Gly Thr Thr Ile Tyr Lys Asn Ile Arg His Gly 515 520 525 Leu Ile Gly Thr Lys Tyr Glu Asn Glu Ser Glu Asp Lys Val Arg Glu 530 535 540 Leu Ile Glu Asn Ala Ala Lys Met Ala Asn Ala His Asp Phe Ile Thr 545 550 555 560 Ala Leu Pro Glu Gly Tyr Glu Thr Asn Val Gly Gln Arg Gly Phe Leu 565 570 575 Leu Ser Gly Gly Gln Lys Gln Arg Ile Ala Ile Ala Arg Ala Val Val 580 585 590 Ser Asp Pro Lys Ile Leu Leu Leu Asp Glu Ala Thr Ser Ala Leu Asp 595 600 605 Thr Lys Ser Glu Gly Val Val Gln Ala Ala Leu Glu Arg Ala Ala Glu 610 615 620 Gly Arg Thr Thr Ile Val Ile Ala His Arg Leu Ser Thr Ile Lys Thr 625 630 635 640 Ala His Asn Ile Val Val Leu Val Asn Gly Lys Ile Ala Glu Gln Gly 645 650 655 Thr His Asp Glu Leu Val Asp Arg Gly Gly Ala Tyr Arg Lys Leu Val 660 665 670 Glu Ala Gln Arg Ile Asn Glu Gln Lys Glu Ala Asp Ala Leu Glu Asp 675 680 685 Ala Asp Ala Glu Asp Leu Thr Asn Ala Asp Ile Ala Lys Ile Lys Thr 690 695 700 Ala Ser Ser Ala Ser Ser Asp Leu Asp Gly Lys Pro Thr Thr Ile Asp 705 710 715 720 Arg Thr Gly Thr His Lys Ser Val Ser Ser Ala Ile Leu Ser Lys Arg 725 730 735 Pro Pro Glu Thr Thr Pro Lys Tyr Ser Leu Trp Thr Leu Leu Lys Phe 740 745 750 Val Ala Ser Phe Asn Arg Pro Glu Ile Pro Tyr Met Leu Ile Gly Leu 755 760 765 Val Phe Ser Val Leu Ala Gly Gly Gly Gln Pro Thr Gln Ala Val Leu 770 775 780 Tyr Ala Lys Ala Ile Ser Thr Leu Ser Leu Pro Glu Ser Gln Tyr Ser 785 790 795 800 Lys Leu Arg His Asp Ala Asp Phe Trp Ser Leu Met Phe Phe Val Val 805 810 815 Gly Ile Ile Gln Phe Ile Thr Gln Ser Thr Asn Gly Ala Ala Phe Ala 820 825 830 Val Cys Ser Glu Arg Leu Ile Arg Arg Ala Arg Ser Thr Ala Phe Arg 835 840 845 Thr Ile Leu Arg Gln Asp Ile Ala Phe Phe Asp Lys Glu Glu Asn Ser 850 855 860 Thr Gly Ala Leu Thr Ser Phe Leu Ser Thr Glu Thr Lys His Leu Ser 865 870 875 880 Gly Val Ser Gly Val Thr Leu Gly Thr Ile Leu Met Thr Ser Thr Thr 885 890 895 Leu Gly Ala Ala Ile Ile Ile Ala Leu Ala Ile Gly Trp Lys Leu Ala 900 905 910 Leu Val Cys Ile Ser Val Val Pro Val Leu Leu Ala Cys Gly Phe Tyr 915 920 925 Arg Phe Tyr Met Leu Ala Gln Phe Gln Ser Arg Ser Lys Leu Ala Tyr 930 935 940 Glu Gly Ser Ala Asn Phe Ala Cys Glu Ala Thr Ser Ser Ile Arg Thr 945 950 955 960 Val Ala Ser Leu Thr Arg Glu Arg Asp Val Trp Glu Ile Tyr His Ala 965 970 975 Gln Leu Asp Ala Gln Gly Arg Thr Ser Leu Ile Ser Val Leu Arg Ser 980 985 990 Ser Leu Leu Tyr Ala Ser Ser Gln Ala Leu Val Phe Phe Cys Val Ala 995 1000 1005 Leu Gly Phe Trp Tyr Gly Gly Thr Leu Leu Gly His His Glu Tyr Asp 1010 1015 1020 Ile Phe Arg Phe Phe Val Cys Phe Ser Glu Ile Leu Phe Gly Ala Gln 1025 1030 1035 1040 Ser Ala Gly Thr Val Phe Ser Phe Ala Pro Asp Met Gly Lys Ala Lys 1045 1050 1055 Asn Ala Ala Ala Glu Phe Arg Arg Leu Phe Asp Arg Lys Pro Gln Ile 1060 1065 1070 Asp Asn Trp Ser Glu Glu Gly Glu Lys Leu Glu Thr Val Glu Gly Glu 1075 1080 1085 Ile Glu Phe Arg Asn Val His Phe Arg Tyr Pro Thr Arg Pro Glu Gln 1090 1095 1100 Pro Val Leu Arg Gly Leu Asp Leu Thr Val Lys Pro Gly Gln Tyr Val 1105 1110 1115 1120 Ala Leu Val Gly Pro Ser Gly Cys Gly Lys Ser Thr Thr Ile Ala Leu 1125 1130 1135 Leu Glu Arg Phe Tyr Asp Ala Ile Ala Gly Ser Ile Leu Val Asp Gly 1140 1145 1150 Lys Asp Ile Ser Lys Leu Asn Ile Asn Ser Tyr Arg Ser Phe Leu Ser 1155 1160 1165 Leu Val Ser Gln Glu Pro Thr Leu Tyr Gln Gly Thr Ile Lys Glu Asn 1170 1175 1180 Ile Leu Leu Gly Ile Val Glu Asp Asp Val Pro Glu Glu Phe Leu Ile 1185 1190 1195 1200 Lys Ala Cys Lys Asp Ala Asn Ile Tyr Asp Phe Ile Met Ser Leu Pro 1205 1210 1215 Glu Gly Phe Asn Thr Val Val Gly Ser Lys Gly Gly Met Leu Ser Gly 1220 1225 1230 Gly Gln Lys Gln Arg Val Ala Ile Ala Arg Ala Leu Leu Arg Asp Pro 1235 1240 1245 Lys Ile Leu Leu Leu Asp Glu Ala Thr Ser Ala Leu Asp Ser Glu Ser 1250 1255 1260 Glu Lys Val Val Gln Ala Ala Leu Asp Ala Ala Ala Arg Gly Arg Thr 1265 1270 1275 1280 Thr Ile Ala Val Ala His Arg Leu Ser Thr Ile Gln Lys Ala Asp Val 1285 1290 1295 Ile Tyr Val Phe Asp Gln Gly Lys Ile Val Glu Ser Gly Thr His Ser 1300 1305 1310 Glu Leu Val Gln Lys Lys Gly Arg Tyr Tyr Glu Leu Val Asn Leu Gln 1315 1320 1325 Ser Leu Gly Lys Gly His 1330 4002 base pairs nucleic acid single linear mRNA NO NO 3 AUGUCCCCGC UAGAGACAAA UCCCCUUUCG CCAGAGACUG CUAUGCGCGA ACCUGCUGAG 60 ACUUCAACGA CGGAGGAGCA AGCUUCUACA CCACACGCUG CGGACGAGAA GAAAAUCCUC 120 AGCGACCUCU CGGCUCCAUC UAGUACUACA GCAACCCCCG CAGACAAGGA GCACCGUCCU 180 AAAUCGUCGU CCAGCAAUAA UGCGGUCUCG GUCAACGAAG UCGAUGCGCU UAUUGCGCAC 240 CUGCCAGAAG ACGAGAGGCA GGUCUUGAAG ACGCAGCUGG AGGAGAUCAA AGUAAACAUC 300 UCCUUCUUCG GUCUCUGGCG GUAUGCAACA AAGAUGGAUA UACUUAUCAU GGUAAUCAGU 360 ACAAUCUGUG CCAUUGCUGC CGCGUCGACU UUCCAGAGGA UAAUGUUAUA UCAAAUCUCG 420 UACGACGAGU UCUAUGAUGA AUUGACCAAG AACGUACUGU ACUUCGUAUA CCUCGGUAUC 480 GGCGAGUUUG UCACUGUCUA UGUUAGUACU GUUGGCUUCA UCUAUACCGG AGAACACGCC 540 ACGCAGAAGA UCCGCGAGUA UUACCUUGAG UCUAUCCUGC GCCAGAACAU UGGCUAUUUU 600 GAUAAACUCG GUGCCGGGGA AGUGACCACC CGUAUAACAG CCGAUACAAA CCUUAUCCAG 660 GAUGGCAUUU CGGAGAAGGU CGGUCUCACU UUGACUGCCC UGGCGACAUU CGUGACAGCA 720 UUCAUUAUCG CCUACGUCAA AUACUGGAAG UUGGCUCUAA UUUGCAGCUC AACAAUUGUG 780 GCCCUCGUUC UCACCAUGGG CGGUGGUUCU CAGUUUAUCA UCAAGUACAG CAAAAAGUCG 840 CUUGACAGCU ACGGUGCAGG CGGCACUGUU GCGGAAGAGG UCAUCAGCUC CAUCAGAAAU 900 GCCACAGCGU UUGGCACCCA AGACAAGCUU GCGAAGCAGU AUGAGGUCCA CUUAGACGAA 960 GCUGAGAAAU GGGGAACAAA GAACCAGAUU GUCAUGGGUU UCAUGAUUGG CGCCAUGUUU 1020 GGCCUUAUGU ACUCGAACUA CGGUCUUGGC UUCUGGAUGG GUUCUCGUUU CCUGGUAGAU 1080 GGUGCAGUCG AUGUGGGUGA UAUUCUCACA GUUCUCAUGG CCAUCUUGAU CGGAUCGUUC 1140 UCCUUGGGGA ACGUUAGUCC AAAUGCUCAA GCAUUUACAA ACGCUGUGGC CGCGGCCGCA 1200 AAGAUAUUUG GAACGAUCGA UCGCCAGUCC CCAUUAGAUC CAUAUUCGAA CGAAGGGAAG 1260 ACGCUCGACC AUUUUGAGGG CCACAUUGAG UUACGCAAUG UCAAGCAUAU UUACCCAUCU 1320 AGACCCGAGG UCACCGUCAU GGAGGAUGUU UCUCUGUCAA UGCCCGCUGG AAAAACAACC 1380 GCUUUAGUCG GCCCCUCUGG CUCUGGAAAA AGUACGGUGG UCGGCUUGGU UGAGCGAUUC 1440 UACAUGCCUG UUCGCGGUAC GGUUUUGCUG GAUGGCCAUG ACAUCAAGGA CCUCAAUCUC 1500 CGCUGGCUUC GCCAACAGAU CUCUUUGGUU AGCCAGGAGC CUGUUCUUUU UGGCACGACG 1560 AUUUAUAAGA AUAUUAGGCA CGGUCUCAUC GGCACAAAGU ACGAGAAUGA AUCCGAGGAU 1620 AAGGUCCGGG AACUCAUCGA GAACGCGGCA AAAAUGGCGA AUGCUCAUGA CUUUAUUACU 1680 GCCUUGCCUG AAGGUUAUGA GACCAAUGUU GGGCAGCGUG GCUUUCUCCU UUCAGGUGGC 1740 CAGAAACAGC GCAUUGCAAU CGCCCGUGCC GUUGUUAGUG ACCCAAAAAU CCUGCUCCUG 1800 GAUGAAGCUA CUUCGGCCUU GGACACAAAA UCCGAAGGCG UGGUUCAAGC AGCUUUGGAG 1860 AGGGCAGCUG AAGGCCGAAC UACUAUUGUG AUCGCUCAUC GCCUUUCCAC GAUCAAAACG 1920 GCGCACAACA UUGUGGUUCU GGUCAAUGGC AAAAUUGCUG AACAAGGAAC UCACGAUGAA 1980 UUGGUUGACC GCGGAGGCGC UUAUCGCAAA CUUGUGGAGG CUCAACGUAU CAAUGAACAG 2040 AAGGAAGCUG ACGCCUUGGA GGACGCCGAC GCUGAGGAUC UCACGAAUGC AGAUAUUGCC 2100 AAAAUCAAAA CUGCGUCAAG CGCAUCAUCC GAUCUCGACG GAAAACCCAC AACCAUUGAC 2160 CGCACGGGCA CCCACAAGUC UGUUUCCAGC GCGAUUCUUU CUAAAAGACC CCCCGAAACA 2220 ACUCCGAAAU ACUCAUUAUG GACGCUGCUC AAAUUUGUUG CUUCCUUCAA CCGCCCUGAA 2280 AUCCCGUACA UGCUCAUCGG UCUUGUCUUC UCAGUGUUAG CUGGUGGUGG CCAACCCACG 2340 CAAGCAGUGC UAUAUGCUAA AGCCAUCAGC ACACUCUCGC UCCCAGAAUC ACAAUAUAGC 2400 AAGCUUCGAC AUGAUGCGGA UUUCUGGUCA UUGAUGUUCU UCGUGGUUGG UAUCAUUCAG 2460 UUUAUCACGC AGUCAACCAA UGGUGCUGCA UUUGCCGUAU GCUCCGAGAG ACUUAUUCGU 2520 CGCGCGAGAA GCACUGCCUU UCGGACGAUA CUCCGUCAAG ACAUUGCUUU CUUUGACAAG 2580 GAAGAGAAUA GCACCGGCGC UCUGACCUCU UUCCUGUCCA CCGAGACGAA GCAUCUCUCC 2640 GGUGUUAGCG GUGUGACUCU AGGCACGAUC UUGAUGACCU CCACGACCCU AGGAGCGGCU 2700 AUCAUUAUUG CCCUGGCGAU UGGGUGGAAA UUGGCCUUAG UUUGUAUCUC GGUUGUGCCG 2760 GUUCUCCUGG CAUGCGGUUU CUACCGAUUC UAUAUGCUAG CCCAGUUUCA AUCACGCUCC 2820 AAGCUUGCUU AUGAGGGAUC UGCAAACUUU GCUUGCGAGG CUACAUCGUC UAUCCGCACA 2880 GUUGCGUCAU UAACCCGGGA AAGGGAUGUC UGGGAGAUUU ACCAUGCCCA GCUUGACGCA 2940 CAAGGCAGGA CCAGUCUAAU CUCUGUCUUG AGGUCAUCCC UGUUAUAUGC GUCGUCGCAG 3000 GCACUUGUUU UCUUCUGCGU UGCGCUCGGG UUUUGGUACG GAGGGACACU UCUUGGUCAC 3060 CACGAGUAUG ACAUUUUCCG CUUCUUUGUU UGUUUCUCCG AGAUUCUCUU UGGUGCUCAA 3120 UCCGCGGGCA CCGUCUUUUC CUUUGCACCA GACAUGGGCA AGGCGAAGAA UGCGGCCGCC 3180 GAAUUCCGAC GACUGUUCGA CCGAAAGCCA CAAAUUGAUA ACUGGUCUGA AGAGGGCGAG 3240 AAGCUCGAAA CGGUGGAAGG UGAAAUCGAA UUUAGGAACG UGCACUUCAG AUACCCGACC 3300 CGCCCAGAAC AGCCUGUCCU GCGCGGCUUG GACCUGACCG UGAAGCCUGG ACAAUAUGUU 3360 GCGCUUGUCG GACCCAGCGG UUGUGGCAAG AGUACCACCA UUGCAUUGCU UGAGCGCUUU 3420 UACGAUGCGA UUGCCGGGUC CAUCCUUGUU GAUGGGAAGG ACAUAAGUAA ACUAAAUAUC 3480 AACUCCUACC GCAGCUUUCU GUCACUGGUC AGCCAGGAGC CGACACUGUA CCAGGGCACC 3540 AUCAAGGAAA ACAUCUUACU UGGUAUUGUC GAAGAUGACG UACCGGAAGA AUUCUUGAUU 3600 AAGGCUUGCA AGGACGCUAA UAUCUACGAC UUCAUCAUGU CGCUCCCGGA GGGCUUUAAU 3660 ACAGUUGUUG GCAGCAAGGG AGGCAUGUUG UCUGGCGGCC AAAAGCAACG UGUGGCCAUU 3720 GCCCGAGCCC UUCUUCGGGA UCCCAAAAUC CUUCUUCUCG AUGAAGCGAC GUCAGCCCUC 3780 GACUCCGAGU CAGAAAAGGU CGUCCAGGCG GCUUUGGAUG CCGCUGCCCG AGGCCGAACC 3840 ACAAUCGCCG UUGCACACCG ACUCAGCACG AUUCAAAAGG CGGACGUUAU CUAUGUUUUC 3900 GACCAAGGCA AGAUCGUCGA AAGCGGAACG CACAGCGAAC UGGUCCAGAA AAAGGGCCGG 3960 UACUACGAGC UGGUCAACUU GCAGAGCUUG GGCAAGGGCC AU 4002 

We claim:
 1. A DNA compound that comprises an isolated DNA sequence encoding SEQ ID NO:
 2. 2. The DNA compound of claim 1 which comprises the isolated DNA sequence which is SEQ ID NO:
 1. 3. A vector comprising an isolated DNA sequence of claim
 1. 4. A vector comprising an isolated DNA sequence of claim
 2. 5. A method for constructing a transformed host cell capable of expressing SEQ ID NO: 2, said method comprising transforming a host cell with a recombinant DNA vector that comprises an isolated DNA sequence of claim
 1. 6. A method for expressing SEQ ID NO: 2 in a transformed host cell said method comprising culturing said transformed host cell of claim 5 under conditions suitable for gene expression.
 7. An isolated DNA molecule of claim 1 or a portion thereof, which is labeled with a detectable moiety.
 8. A host cell containing the vector of claim
 3. 9. A host cell containing the vector of claim
 4. 10. A method for determining the fungal MDR inhibition activity of a compound which comprises: a) placing a culture of fungal cells, transformed with a vector capable of expressing atrD, in the presence of: (i) an antifungal agent to which said fungal cell is resistant, but to which said fungal cell is sensitive in its untransformed state; (ii) a compound suspected of possessing Aspergillus nidulans MDR inhibition activity; and b) determining the fungal MDR inhibition activity of said compound by measuring the ability of the antifungal agent to inhibit the growth of said fungal cell.
 11. A method of claim 10 wherein the fungal cell is Saccharomyces cerevisiae.
 12. The protein of SEQ ID No. 2 in purified form.
 13. A strain of A. nidulans wherein said strain carries a gene disruption or gene replacement at the atrD locus such that said strain does not produce the atrD protein product.
 14. A method for identifying an antifungal compound comprising the steps of: a. culturing in the presence of a test compound a strain of claim 13; b. culturing said strain in the absence of said test compound; and c. comparing the growth of said strain in step (a) with the growth in step (b). 